Separating materials for chromatography and electrophoresis applications comprising regiodefined functionalised cyclodextrins chemically bonded to a support via urethane functionalities

ABSTRACT

Novel and improved chiral stationary phases (CSP) materials comprising a support and completely regiodefined derivitised cyclodextrin chemically bonded via single or double urethane linkage(s) universally applicable in HPLC, LC, TLC, and CCE are obtained using a process based on the almost quantitative reaction of pre-synthesized regiodefined per-functionalized mono- or di- azidocyclodextrin with primary amines.

This application is a divisional of application Ser. No. 09/140,743,filed on Aug. 26, 1998, now U.S. Pat. No. 6,017,458, the entire contentsof which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the development of novel separatingmaterials for high performance liquid chromatography (HPLC), liquidchromatography (LC), thin layer chromatography (TLC), capillaryelectrophoretic chromatography (CCE) and counter-current chromatographicprocesses, which essentially comprise amine-bearing support materialsand functionalised cyclodextrins (CD) chemically bonded via a urethanelinkage. More significantly, our procedure affords materials in whichthe cyclodextrins are bonded to the support with well defined chemicalstructure and good experimental reproducibility.

BACKGROUND OF THE INVENTION

The applicability of cyclodextrins in chromatographic separation andpurification processes were previously described at length in reviews byW. L. Hinze, Separation and Purification Methods, 1981, 10(2), 159-237;Y. Kawaguchi, et al., Anal. Chem., 1983, 55, 1852; D. M. Armstrong, etal., Anal. Chem., 1985, 57, 234 and S. Li, et al., Chem. Rev., 1992, 92,1457. Chromatographic separation on chiral stationary phases (CSP) isalso the most convenient analytical method for the determination ofenantiomeric purity (see for example S. G. Allenmark, ChromatographicEnantioseparations: Methods and Applications, 2nd ed., Prentice Hall,N.J., 1991). In recent years, tremendous research efforts were made inbonding cyclodextrins to solid matrices, such as silica gel, via aminoor amido linkages. However, these bonds were inherently unstable tohydrolysis, thus placing severe limitations on their use in aqueousmedia. Alternative approaches for immobilizing CD using hydrolyticallymore stable ether linkages (U.S. Pat. No. 4,539,399) or carbamic acidmoieties (U.S. Pat. No. 503,898) were also investigated. However, in allthese approaches, arising from the presence of multiple hydroxy moietiesin the CD starting materials, regioselective derivatisation ofcyclodextrin cannot be readily effected. Thus, reaction may take placeon the 2, 3 or 6- position of cyclodextrin and may result in mixtures ofmulti-functionalised CDs instead of the desired regiodefined compound.

It was often reported that derivatized CD stationary phases showdefinite enantioselectivity for a variety of compounds while pristinecyclodextrin bonded LC stationary phases depict low enantioselectivity.Thus, as an example, enantioselectivity of the materials were generallyincreased with increasing degree of derivatisation of the —OH groups onCD with carbamate groups on cyclodextrin as of an increasing surfaceconcentration of the functionalised cyclodextrin immobilized on thesupport materials (D. W. Armstrong et al., Anal Chem., 1990, 62, 1610;T. Hargitai et al., J. Chromatogr., 1993, 628, 11; T. Hargitai, et al.,J. Liq. Chromatogr., 1993, 16(4), 843). In order to maximise the extentof cyclodextrin derivatisation, large molar excesses of derivatizingreagents under vigorous conditions were often used. However, as thederivatisation processes invariably involved the prior immobilization ofunderivatised cyclodextrin on the support material followed byfunctionalisation procedures involving solid-liquid phases, partialderivatisation of the hydroxyl groups of the cyclodextrin usuallyresulted with large, sterically encumbered substituents consistentlyhaving a lower extent of derivitisation. In addition, these methods didnot give good reproducibility or uniformity of product with theconsequence that separation of enantiomers may vary from batch to batchof the obtained CD-based CSP.

OBJECTIVES AND BRIEF SUMMARY OF THE INVENTION

One objective of this invention is the obtainment of novel and improvedCSP materials comprising a support and completely regiodefinedderivatised cyclodextrin chemically bonded via single or double urethanelinkage(s), universally applicable in HPLC, LC, TLC and CCE. Applicationin counter-current chromatographic processes would thus afford a viableand efficient means into bulk/ industrial scale enantioseparation, whichwould be of interest particularly to pharmaceutical firms involved inenantioseparation of racemic chiral drugs.

Another objective of the invention is to derive methodologies for thepreparation of materials mentioned above. These and other objectiveswill become apparent or will be highlighted in the ensuing description.

The present invention is based on the almost quantitative reaction ofpre-synthesized regiodefined perfuctionalized monoazidocyclodextrin withprimary amines based on an extended application of the Staudingerreaction which we have investigated. In addition, application ofexisting synthetic strategies (G. Wenz, Angew. Chem. Int. Ed. Eng.,1994, 33, 803 and references therein) into regiodefined disulphonatedCDs, often referred to as capped cyclodextrins, can likewise affordregiodefined diazido perfunctionalised CDs suitable for reaction withamines. When aminized silica gel was used in place of the primary amineunder similar conditions, the perfunctionalized mono- (ordi-)azidocyclodextrin could be immobilized onto the surface of silicagel easily via stable urethane linkage(s), usually adopted inPirkle-typed or protein-based CSPs (N.Oi, et al., J. Chromatogr., 1983,111, 257; W. H. Pirkle, J. Chromatogr., 1985, 322, 295; W. H. Pirkle, J.Liq. Chromatogr., 1986, 9, 443; C. J. Welch, J. Chromatogr. A, 1994,666, 3; W. H. Pirkle, et al., J. Am. Chem. Soc., 1989, 111, 9222; A. M.Dyas, Recent Advances in Chiral Separations, Plenum Press, New York,1991). Furthermore, when perfunctionalized mono- or diazidocyclodextrinsare reduced into the corresponding perfunctionalized mono- or diaminocyclodextrins, the latter can be easily anchored onto the surface of thesupport by suitable coupling reagents via urethane or amido linkages.

The invention therefore relates to a separating material used inchromatography, essentially comprising a support material andregiodefined perfunctionalized cyclodextrins chemically bonded to thissupport and characterized by a bonding via single or double urethanemoieties. The invention also relates to a process for the production ofthis separating material in which the perfunctionalized mono-(ordi)azidocyclodextrin is:

a. coupled directly to any support which is carrying free —NH₂ groups onthe surface of the support.

b. reacted with any alkenyl amines to give the correspondingperfunctionalized cyclodextrin with mono- (or di)alkenyl substitutedside chain(s) via urethane linkage(s) and then allowing this derivativeto be hydrosilylized with HSiR_(n)X_(3−n)(where R and X are alkyl,alkyloxy, aryl or halide) and thereafter effecting an immobilizationonto the surface of a support material.

c. reacted with any aminosilanes containing at least one furtherreactive group to afford the corresponding cyclodextrin-silanederivative. The resulting cyclodextrin-silane derivative is treated withsilica gel to afford the perfunctionalized cyclodextrin bonded silicagel.

d. reduced to perfunctionalized mono- or diaminocyclodextrin and thenanchored onto the surface of a support via coupling reagents.

Support which can be employed are silica gel or any alternativeinorganic materials such as Al₂O₃, TiO₂ or ZiO₂ or synthetic polymersupports, preferably incorporating existing free NH₂ group on thesurface. The support employed of choice is silica gel, which iscommercially available in a wide range of different shapes and sizes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a representative completely substituted cyclodextrin(β-cyclodextrin) immobilized onto the surface of a support by a urethanelinkage.

FIG. 2 depicts the FT-IR spectra of the materials obtained by presentinvention with different substituted group, which prove that theperfunctionalized cyclodextrins have already been anchored to thesurface of the silica gel.

FIGS. 3A, 3B, and 3C depict the representative HPLC enantioseparation ofracemic β-blockers propranolol, oxprenolol, and alprenolol using theproduct of example 3.

DETAILED DESCRIPTION OF THE INVENTION

The CSP according to the invention can be produced by four differentapproaches as in a. to d. above. From a. to c., the key step in allvariety is the same, involving reacting perfunctionalized mono- (ordi)azidocyclodextrin with free amines via a facile one-pot procedure inhigh yield or good immobilization contents. The cyclodextrin used may beof any of the α, β or γ form or combination thereof whilst thederivatisation substituent groups may be alkyl, aryl, ester andcarbamate.

In method a., the perfunctionalized mono- (or di-)azidocyclodextrin iscoupled directly onto a support which has already been modified withamino groups. This —NH₂ bearing material can either be obtainedcommercially in finished form or prepared by a silylation reaction ofthe support with an appropriate aminosilane. For this purpose,particular preference is made to aminosilane such as, for example,3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane,3-(2-aminoethyl)aminopropyltriethoxysilane and3-(2-aminoethyl)aminopropyltrimethoxy silane.

In the method b., suitable alkenyl amines may be, but not limited to theform: CH₂═CH(R₁)_(m)(R₂)_(n)NH₂ where m, n=1-20.

In the method c., the aminosilanes described in method a. are suitablefor this purpose. Here too, urethane linkage(s) is formed between theNH₂ groups of the support and the perfunctionalized cyclodextrin.

In the method d., the coupling reagents may be OCN—R—NCO, ClOC—R—COCl orSiX_(n)Y_(3-n)—R—NCO, where X and Y are alkyl, alkyloxy, aryl or halide.

The connecting spacer between the support and the perfunctionalizedcyclodextrin may comprise a relative short or relative long chain. Thischain preferably has 3-20 atoms and may contain nitrogen, oxygen orsilicon atoms in addition to carbon atoms.

The material obtained according to the invention can, if desired befurther subjected to an “end-capping” reaction. In this, the remaininghydroxyl groups on the silica gel surface are reacted in a known mannerwith a reactive silane, such as, for example, trimethylchlorosilane oralternatively hexamethyldisilazane, in order to comnplete the blockingof the surface hydroxyl groups.

Above all, by present invention, the derivatised cyclodextrin can beimmobilized onto the surface of the support via a single (or double)urethane linkage(s). In the CSP material obtained by the presentinvention, the chemical structure of cyclodextrin is well defined andthe immobilisation can be effected with good reproducibility.

EXAMPLES

The following examples illustrate the practice of the present invention.

Cyclodextrin monofunctionalized with an azido moiety at the 6-positionwas prepared using the previously reported procedure by L. D. Melton etal., Carbohydr. Res., 1971, 18, 29; H. Parrot-lopez, et al, TetrahedronLett., 1992, 33, 209; R. C. Petter, J. Am. Chem. Soc., 1990, 112, 3860.Cyclodextrin difunctionalized with azido moieties at the 6-position wasprepared according to the reported method by I. Tabushi, et al.,Tetrahedron Lett., 1977, 18, 1527; I. Tabushi, et al., J. Am. Chem.Soc., 1984, 106, 5267; G. L. Yi, et al., J. Org. Chem., 1993, 58, 2561.The other hydroxyl groups of the 6-mono (or di-)azido substitutedcyclodextrin can subsequently be completely derivatised with differentsubstituents groups such as alkyl, aryl, ester and carbamate.

Peracetylated-monoazido-β-cyclodextrin was prepared easily by stirring6-monoazido-β-cyclodextrin with (AcO)₂O/Pyridine at 40° C. in 90% yield.

Pernethylated-monoazido-β-cyclodextrin was prepared by reaction of6-monoazido-β-cyclodextrin in CH₃I/DMF/NaH at 40° C. in 70% yield.

Monoazido-β-cyclodextrin perphenylcarbamate was prepared by reaction of6-monoazido-β-cyclodextrin in PhNCO/Pyridine at 80° C. in 70% yield.

Aminized silica-gel was prepared according to the literature method byT. Hargital et al., J. chromatogr., 1993, 628, 11, with the followingcomposition as determined from elemental analysis: C% 3.25, H% 0.96, N%0.98.

Example 1

4 g of animized silica gel was stirred in 30 ml ahydrous THF into whicha continuous stream of CO₂ gas was passed. After 20 mins, 1.2 g ofperacetylated monoazido-β-cyclodextrin in 10 ml anhydrous THF was added.Stirring was continued another 5 mins, whence 0.3 g oftriphenylphosphine in 10 ml anhydrous THF was added. The mixture wasstirred 10 hrs with constant bubbling of CO₂ at room temperature. Thereaction mixture was then transferred to a soxhlet extraction apparatusand extracted with acetone for 24 hrs. After removal of the acetone invacuo, the peracetylated cyclodextrin immobilized silica gel wasobtained having the following composition as determined from elementalanalysis: C% 8.89, H% 1.78, N% 0.80.

Examples 2 and 3

Example 1 was repeated using monoazide-permethyl-β-cyclodextrin ormonoazido-β-cyclodextrin perphenylcarbamate in place of theperacetylated monoazido-β-cyclodextrin of example 1. The elementalanalyses were as follow:

1. Bonded with permethylated-β-cyclodextrin: C% 8.82, H% 1.88, N% 0.94.

2. Bonded with β-cyclodextrin perphenylcarbamate: C% 11.32, H% 2.0, N%1.24.

Example 4

A solution of the 10-undecenylamine (2.0 mmol) in 10 ml dry THF wassaturated with dry CO₂ at room temperature with stirring, immediatelyprecipitate of ammonium carbamate derivative was observed. Withcontinuous bubbling of CO₂ into the suspension, monoazide peracetylβ-cyclodextrin(1.80 mmol) in 10 ml anhydrous THF was added in a singleportion, followed by addition of PPh₃(1.80 mmol) in 10 ml dry THF. Thiswas allowed to react for about 5 hrs, TLC revealed that no startingmaterials existed. After evaporation to dryness, the product waspurified by column chromatography with ethyl acetate-acetone(1:1) aseluant in 97% yield.

Mp:115-117° C., [α]=+106.1°(c 1.0 in CHCl₃); IR: 3427 (N-H), 2937, 2859(C-H), 1744, 1663 (C═O); ¹H NMR (CDCl₃) δ(ppm): 5.89-5.76 (m,1H),5.38-5.21 (m, 7H), 5.16-4.94(m, 11H), 4.84-4.68 (m,7H), 4.58-4.50 (m,6H), 4.35-4.26 (m, 6H), 4.20-4.05 (m, 7H), 3.78-3.66 (m, 7H), 3.57-3.51(m, 1H), 3.49-3.32 (m, 1H), 3.30-3.16 (m,1H), 3.11-3.00 (m, 1H),2.18-2.00 (several s, 60H), 1.46-1.19 (m, 16H); ¹³C NMR (CDCl₃, 25° C.)δ (ppm):170.6-169.3, 158.0, 139.1, 113.9, 96.6-96.4, 78.3-76.2,70.7-69.5, 62.4, 41.2, 40.3, 33.7-26.8, 20.6-20.4; Elemental analysiscalcd for C₉₄H₁₃₂N₂O₅₅ (2168.76): C 52.01%, H 6.13%, N 1.29%; Found C51.72%, H 6.30%, N 1.20%.

Examples 5 and 6

Example 4 was repeated using monoazido-permethyl-β-cyclodextrin ormonoazido-β-cyclodextrin perphenylcarbamate in place of themonoazido-peracetyl-β-cyclodextrin of example 4 with basic propertiesshown as following:

6-(10′-undecenyl)urea-permethyl-β-cyclodextrin: yield 97%, Mp: 72-74° C.[α]=+132.90° (c 1.0, CHCl₃);

6-(10′-undecenyl)urea perphenylcarbarmate-β-cyclodextrin: yield 95%, Mp:198-200 ° C. [α]=+8.5° (C1.0, CHCl₃);

Example 7

1.5 g of product obtained in Example 4 was stirred with 5 mltriethoxysilane and 10 mg of tetrakls(triphenylphosphine)platinum at 60°C. After 24 hrs the mixture was adsorbed with 2 cm high silica gel in aBuchner funnel and washed with 100 ml ether, after removal of the etherand volatile by vacuum, the residue was dissolved in 50 ml anhydroustoluene, 4.0 g of silica gel(which had already been dried over vacuum at120° C. for) was added, the mixture was stirred at 80° C. for 8 hrs.After filtration and extracted in soxhlet apparatus with acetone for 24hrs, the product was obtained with the element analysis as shownfollowing:

C% 6.98, H% 1.52,N% 0.10

Examples 8 and 9

Example 7 was repeated using monoazido-permethylated-β-cyclodextrin ormonoazido-β-cyclodextrin perphenylcarbamate in place of theperacetylated monoazido-β-cyclodextrin of example 7 with elementalanalyses shown as following:

1. Bonded with permethylated-β-cyclodextrin: C% 6.50, H% 1.32, N% 0.10.

2. Bonded with β-cyclodextrin perphenylcarbarnate: C% 9.40, H% 1.80, N%0.83.

It will be noted that in the products produced by the present invention,the cyclodextrin is completely derivatised with the exception of thesingle (or double) 6-glucosidic position(s) connected to the supportmaterial via urethane linkage(s) which is (are) very stable andfrequently adopted in Pirkle-typed and Protein-based CSPs. Such completemodification of cyclodextrin affords a regiodefined chemical structureand have good experimental reproducibility.

Having now described the invention, it is not intended that it belimited except as may be required by the appended claims. Theembodiments of the invention in which an exclusive property of theprivilege is claimed are defined in the following claims.

What is claimed is:
 1. A method for chromatographic or electrophoreticseparation of a mixture comprising, respectively, contacting a samplecomprising said mixture with or applying a sample comprising saidmixture to a separation support material, said separation supportmaterial being one produced by a process comprising any one of thefollowing steps: (a) reacting a regiodefined mono-azidoperfunctionalized cyclodextrin or a regiodefined di-azidoperfunctionalized cyclodextrin with a support that carries free NH2groups; (b) reacting a regiodefined mono-azido perfunctionalizedcyclodextrin or a regiodefined di-azido perfunctionalizedcyclodextrinwith an alkenyl amine to produce a product comprising a C═Cgroup linked to a side chain via a urethane linkage, furtherhydrosilating the C═C group of said product with an active silane, andchemically immobilizing the product onto the surface of a support; (c)reacting a regiodefined mono-azido perfunctionalized cyclodextrin or aregiodefined di-azido perfunctionalized cyclodextrin with an aminosilanecontaining at least one further reactive group to form a product, andchemically immobilizing the product onto a surface of a support; and (d)reducing a regiodefined mono-azido perfunctionalized cyclodextrin or aregiodefined di-azido perfunctionalized cyclodextrin to a monoamino ordiamino perfunctionalized cyclodextrin, and then anchoring the mono- ordiamino perfunctionalized cyclodextrin onto the surface of a support bya coupling reagent, and, in the case of electrophoretic separation,applying an electric field.
 2. The method for separation of claim 1,which comprises method for chromatographic separation of a mixture. 3.The method of claim 2, wherein said mixture comprises a mixture ofenantiomers of a compound.
 4. The method for separation of claim 1,which comprises method for electrophoretic separation of a mixture. 5.The method of claim 4, wherein said mixture comprises a mixture ofenantiomers of a compound.